DPhA-EtOBz-TSC

Colorectal cancer (CRC) remains one of the most prevalent malignancies globally, with limited therapeutic options for advanced-stage patients due to acquired drug resistance and systemic toxicity. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a promising therapeutic target for CRC, while cystathionine γ-lyase (CSE)-a key enzyme in hydrogen sulfide (H₂S) biosynthesis-plays a critical role in maintaining redox homeostasis and suppressing ferroptosis in cancer cells. Here, we synthesized a novel small-molecule compound, DPhA-EtOBz-TSC, and systematically evaluated its anti-CRC efficacy and underlying molecular mechanism. The chemical structure of DPhA-EtOBz-TSC was confirmed by ¹H nuclear magnetic resonance (¹H NMR), ¹³C NMR, and high-resolution mass spectrometry (HRMS). In vitro studies using human CRC cell lines (HCT8 and SW480) demonstrated that DPhA-EtOBz-TSC specifically targeted and inhibited CSE: molecular docking and cellular thermal shift assay (CETSA) confirmed direct binding between DPhA-EtOBz-TSC and CSE (binding affinity ΔG = -29.07 ± 3.85 kcal/mol), while Western blot analysis revealed a concentration-dependent reduction in CSE protein levels in SW480 and HCT8 cells treated with DPhA-EtOBz-TSC. Mechanistically, DPhA-EtOBz-TSC-induced CSE downregulation disrupted the CSE-H₂S-GPX4 axis, leading to reduced glutathione (GSH) levels, decreased glutathione peroxidase 4 (GPX4) activity, and accumulation of lipid reactive oxygen species (ROS) and malondialdehyde (MDA)-hallmarks of ferroptosis. Additionally, Electron microscopy and JC-1 staining revealed that DPhA-EtOBz-TSC induced significant alterations in mitochondrial morphology and a marked reduction in mitochondrial membrane potential (ΔΨm). Functionally, DPhA-EtOBz-TSC exhibited potent in vitro anti-CRC activity with IC₅₀ values of 25.15 μM (HCT8) and 26.11 μM (SW480) respectively, and suppressed migration and invasion. In vivo, DPhA-EtOBz-TSC (10 mg/kg/day, i.p.) significantly suppressed the growth of CT26 cell-derived xenograft tumors in BALB/c mice without causing obvious histopathological abnormalities in major organs (heart, liver, spleen, lung, kidney). Immunohistochemical staining of tumor tissues confirmed increased lipid peroxidation and decreased CSE/GPX4 expression in DPhA-EtOBz-TSC-treated mice, consistent with in vitro findings. In conclusion, DPhA-EtOBz-TSC is a novel CSE-targeting compound that induces ferroptosis in CRC cells and inhibits tumor growth in vivo. Our findings identify DPhA-EtOBz-TSC as a potential lead compound for CRC therapy and highlight the CSE-H₂S-GPX4 axis as a viable therapeutic target for ferroptosis-based anticancer strategies.